1. Field of the Invention
The invention relates generally to a system for imaging. In particular, the invention relates to a system for generating three dimensional images of a sample.
2. Background of the Invention
Multiplanar microscope imaging enables parallel computation of autofocus parameter values for high-speed image cytometry. Although image cytometry exhibits many potential advantages over flow cytometry, its substantially slower speed has limited its use to fewer applications. In commercial image cytometry instruments, long scanning times have typically been circumvented by identification of small areas of interest during high speed, low resolution scans for subsequent analysis at high resolution. This two-pass strategy of analyzing only a few cells at high resolution is a disadvantage and often cannot be used at all where dim fluorescence demands higher numerical aperture (NA) objectives. Continuous stage motion synchronized with line array or time-delay-and-integrate (TDI) CCD image acquisition (analogous to web inspection in machine vision) is capable of increasing scan speeds by an order of magnitude or more, but until recently lacked the autofocus required for higher resolution (NA>0.5) objectives where depth of field is about the thickness of a cell monolayer.
In high-resolution scanning cytometry, cell borders and textures are important features necessary for quantitative measurements and classification. To achieve the high level of details, optical systems with high numerical aperture (NA) are required. Objectives with NAs>0.5 often reduce the depth-of-focus to less than 1 micron, requiring refocusing for each field of view (FOV) to maintain image quality. Therefore, autofocus can be essential in scanning cytometry. Currently, most autofocus implementations are for incremental scanning. Because these methods require sequential acquisition of a series of test focus planes and start-and-stop motion of the microscope stage, the scanning rates of these implementations are slow, especially at high magnification. There have been efforts to improve scanning speed by incorporating continuous stage movement with on-the-fly autofocus. Simultaneous multiplanar image acquisition for tracking focus and 3D imaging employing fiber optic bundles optically coupling an array of cameras to a series of imaging planes is disclosed in M. Bravo-Zanoguera and J. H. Price, “Simultaneous Multiplanar Image Acquisition in Light Microscopy,” SPIE 3260, pp. 194-200, 1998. However, optical fibers and optical waveguides are fragile, require mechanically complicated fixtures, and have light transmission efficiency of only about 50%. For simultaneous multiplanar acquisition to be practical and widely implemented, a simpler setup is necessary.
In microscopy, several image criteria including resolution, contrast, and entropy have been used in different methods for determining focus; however, the resolution criterion has dominated in biological microscopy and has been shown to be very robust and accurate. Highest resolution is obtained at best focus. Details blur as the image is defocused and resolution is lost. One measure of resolution is the high-frequency content of the Fourier frequency spectrum. Typical focussing implementations utilize highpass or bandpass filters making up of the first and the second derivatives of the image intensity. More recently, higher order digital and analog filters have been shown to eliminate spurious maxima by removing lower frequency contrast reversals. The resulting frequencies are used to compute a focus index as a function of the axial (z) position (i.e., an autofocus circuit outputs a voltage that is proportional to the degree of sharpness of the image.) Measurement of this focus index at several planes of focus together with an appropriate algorithm is used to calculate the position of best focus.
Simultaneous multiplanar acquisition is based on the use of axial displacement of each detector to focus on corresponding different planes in specimen space. This axial displacement, however, usually results in some change in magnification. Changes in magnification translate to different distributions in the frequency spectrum, which in turn alter the focus index. Previous autofocus implementations have assumed that the effect of magnification on focus index is negligible, or can be corrected by changing the focus index gain. In some cases, the magnification changes are as high as 6% for a 10-micron shift in the specimen plane, depending on the type of microscope and objective used. Furthermore, in future 3D imaging applications, the changes in magnification will require correction before a stack of 2D images is further processed and viewed as a 3D image. In addition, by correcting for difference in magnification before the data is recorded, later image processing tasks such as filtering, segmentation, and classification are simpler and possibly more accurate by not having to compensate for magnification. Thus, magnification correction is an important feature of multiplanar image acquisition.
There are mainly three approaches to autofocus in scanning cytometry: pre-determined focussing, one camera focussing, and multiple camera focussing. Pre-determined focussing is often used in low-resolution and low NA scanning, in which the depth-of-field can be as much as 10 microns. Typically in these applications, foci at three locations are measured and used to determine an average focus plane and the scan of the entire slide is performed with little or no refocusing.
In incremental scanning, focussing is typically performed using one camera. In each FOV, the objective is displaced in small increments to collect a z-series typically of seven to nine 2D images. Sharpness of each image is measured and used to find the in-focus position. With the assumption that an ideal focus function curve has a sharp peak centered at the best focus position, common implementations use either a maximum algorithm or a power-weighted algorithm to determine focus. The maximum algorithm chooses the position of the sharpest test image to be the in-focus position, thus usually requiring more planes for accuracy. The power-weighted algorithm calculates the best focus position by using the focus indices as weights of their respective axial positions. The focus indices are often raised to a power to further increase robustness. Due to the sequential z-series acquisition and start-and-stop stage motion, incremental scanning rate is slow and usually limited to approximately 3-4 Hz.
To achieve faster scanning speed, the stage motion is kept continuous in continuous scanning. This, however, requires autofocus to be done on the fly and the collection of the test focus images to be carried out in parallel. One such method is disclosed in M. Bravo-Zanoguera and J. H. Price, “Simultaneous Multiplanar Image Acquisition in Light Microscopy,” SPIE 3260, pp. 194-200, 1998. Multiple time-delay-and-integrate (TDI) cameras are coupled to different axial positions in the imaging volume by means of fiberoptic imaging bundles (FIB). This continuous scanning setup is complex and fragile. Due to side-by-side arrangements of the FIBs, there are non-uniform spatial delays between the cameras.
Consequently, the autofocus algorithm must keep track of these delays and account for them appropriately in calculating the focus position. In order to accommodate different magnification and fiber arrangements, the FIBs are required to slide against each other axially. The inherent fragility of the FIBs and their bonds to the CCD detectors requires a mechanically complex supporting fixture and extreme care be taken in its operation. Furthermore, the FIBs have low light transmission efficiency at approximately only 50%.
There is a need for a new and simplified optically aligned and magnification matched setup for multiplanar acquisition.